Introducing nucleic acids into living cells is an important process in modern biological research, industry, and medicine. Efficient delivery of a functional nucleic acid into a living cell is an indispensable component of genetic engineering, recombinant protein production, and medical technologies known as gene therapy.
For example, gene therapy involves the transfer of normal, functional genetic material into specific cells to correct an abnormality due to a deficient or defective gene product. A variety of methods have been developed to facilitate both in vivo, in vitro, or ex vivo gene transfer.
Nucleic acid therapies involve the transfer of natural or synthetic oligonucleotides and polynucleotides into normal and/or pathological cells with the purpose of correcting or eliminating the diseased cells. For example, antisense oligonucleotides and interfering RNAs such as siRNAs and shRNAs are used to block undesirable pathways of protein expression in the cells. Plasmids, e.g., plasmids that comprise one or more protein-encoding sequences, may be introduced into cells to correct a cellular defect associated with a defective or absent gene or gene product, or to induce tumor cell death. Polynucleotide inductors of immunity, such as poly(I, C) or oligo- and polynucleotides having methylated GC pairs are used to increase the patients' defense against pathogens such as viruses or cancer cells. Ribozymes are ribonucleic acids that catalyze selective degradation of other polynucleotides in the diseased cells, for example, in cancer or virus-infected cells. Because oligo- and polynucleotides generally have low permeability through cell membranes, and are quickly eliminated from the body, there is the need for oligo/polynucleotide delivery vehicles that would allow enhanced intracellular delivery and protection from degradation and/or elimination from the body.
One useful method for providing nucleic acid therapies is to encapsulate therapeutic nucleic acids into liposomes suitable for administration to a patient. Liposome technology has been developed and commercialized for the delivery of conventional pharmaceutical agents, but to date therapeutic nucleic acid containing liposomes have not been commercialized. To date many publications demonstrate that liposome-plasmid DNA complexes can mediate efficient transient expression of a gene in cultured cells but poor in vivo transfection efficiencies. Unlike viral vector preparations, liposome-nucleic acid complexes can have insufficient stability, and thus can be unsuitable for systemic injection.
The ability of a liposome to deliver a nucleic acid to a cell can be enhanced by adding adequate levels of cationic lipids to the lipid bilayer of the liposome, but cationic lipids are not cell specific. Because many diseases, such as cancers, are limited to specific organs, tissues, or cell types, it is desirable to transfer nucleic acids in an organ, tissue, or cell selective fashion. Immunoliposomes—liposomes comprising exterior antibody functionalities, are capable of achieving such cell selective transfer of nucleic acids to cells. The presence of large amounts of cationic lipids on liposomes a useful for achieving introduction of a therapeutic entity within the liposome into a cell. In immunoliposomes however, amounts of cationic lipids that are effective to achieve such an effect can result in undesirable non-specific binding to cells, which decreases the ability to specifically direct a liposome-nucleic acid complex to a target cell or cell type.
It would be desirable to have improved small, active, and biocompatible liposome-nucleic acid complexes that are capable of being prepared as or converted to immunoliposomes selectively targeted to specific cell types and methods for making and such complexes.